Leishmania (Sauroleishmania) tarentolae isolation and sympatric occurrence with Leishmania (Leishmania) infantum in geckoes, dogs and sand flies

The trypanosomatid protist Leishmania tarentolae is a saurian-associated parasite vectored by the Sergentomyia minuta sand fly. This study aimed to confirm the circulation of L. infantum and L. tarentolae in sand flies, reptiles and dogs and to isolate new strains of these protists. Reptilian and sheltered dog blood samples were collected, and sand flies were captured. Samples were tested for Leishmania spp. using duplex real-time PCR (dqPCR) and real-time PCR (qPCR); the origin of blood meal was identified in engorged sand flies by conventional PCR. The reptilian blood and intestinal content of sand fly females were cultured. Dog sera were tested by IFAT using both Leishmania species. Four Tarentola mauritanica geckoes were molecularly positive for L. infantum or L. tarentolae, with no co-infections; moreover, amastigote-like forms of L. infantum were observed in the bone marrow. 24/294 sand flies scored positive for Leishmania spp. by dqPCR, 21 S. minuta and two Phlebotomus perniciosus were positive for L. tarentolae, while only a single Ph. perniciosus was positive for L. infantum. Blood meal analysis confirmed reptile and dog in S. minuta, dog and human in Ph. perniciosus and dog in Phlebotomus neglectus. Two axenic strains of L. tarentolae were obtained. Twelve of 19 dogs scored positive for L. infantum and L. tarentolae by IFAT and three of them also for L. infantum by dqPCR, and six by qPCR. These data confirm the sympatric circulation of L. infantum and L. tarentolae in geckoes, sand flies, and dogs, and suggest that geckoes may be infected with L. infantum.


Introduction
Leishmaniases are important diseases affecting mammals, including humans, in tropical, subtropical, and temperate regions, with more than 350 million people infected worldwide [1]. The genus Leishmania (Kinetoplastea, Trypanosomatidae), transmitted predominantly by phlebotomine sand flies (Diptera, Psychodidae), includes more than fifty species which infect mammals and reptiles. From those, about twenty parasitize humans, causing cutaneous, mucocutaneous and visceral leishmaniasis [2,3]. In the Mediterranean basin, Leishmania infantum is the main species, causing zoonotic cutaneous and visceral leishmaniasis in humans, and infects more than 2.5 million dogs [4]. In the same area, other pathogenic species of the subgenus Leishmania occur (e.g., Leishmania donovani in Cyprus and Leishmania tropica in Greece), while species typical of reptiles, belonging to the subgenus Sauroleishmania, (e.g., Leishmania chameleonis and Leishmania tarentolae) were found in Algeria, France, and Italy [3,[5][6][7]. Indeed, ecological and anthropic drivers (i.e., climate changes, animal translocation, wildlife movements, or globalization) have amplified the risk of alien Leishmania spp. introduction [8], and the spreading of sand fly populations in new localities results in the northward shift of leishmaniasis [9].
The abovementioned factors pose new challenges to medical and veterinary practitioners, given the possibility of diagnostic inaccuracies or cross-reactivities, such as in the case of the simultaneous occurrence of L. infantum and L. tropica in dogs in Israel [10] and of L. infantum and L. tarentolae in dogs in Italy [7].
In particular, saurian-associated L. tarentolae has been detected in geckoes Tarentola mauritanica and Mediodactylus kotschyi from Italy [11] and Tarentola annularis from Sudan [12]. Recently, this species was molecularly detected in lizards Podarcis filfolensis and Podarcis siculus [7,13]. The species was molecularly detected also in human blood in central Italy [14], in islanders of the Pelagie archipelago [15] and in sheltered dogs in Italy [7]. Mammalian species L. infantum was molecularly detected in lizards (i.e., P. siculus) and geckoes (i.e., T. mauritanica, Hemidactylus turcicus) inhabiting dog shelters in southern Italy [13]. The high abundance of the natural vector Sergentomyia minuta and the detection of human and dog blood in engorged females [14,16] suggest the possibility of mammalian exposure to L. tarentolae. Indeed, both L. infantum and L. tarentolae may infect the same sand fly species where their distribution overlaps. In Italy, this is the case of Phlebotomus perfiliewi and Phlebotomus perniciosus that were molecularly positive for L. tarentolae [7,14] and S. minuta for L. infantum [14,[17][18][19]. In addition, experimental infections demonstrated that L. tarentolae develops in Ph. perniciosus and Phlebotomus papatasi [20], the main vectors of L. infantum and Leishmania major, respectively. Although scientific interest on L. tarentolae is continually growing, its life cycle, pathogenicity, tropism and overall biology remain largely unknown, despite many isolations and molecular characterization efforts [11,[20][21][22][23]. The obtained strains have been of great importance for the development of biotechnological tools, given by the production of cultured promastigotes at the industrial level [2,24]. In addition, two isolates are available commercially in culture, namely, TarII (ATCC: 30267) (Algeria, 1939; [21]) and LEM125 (France, 1981; [25]) strains, both obtained from T. mauritanica geckoes. Importantly, LEM125 strains can be transiently infectious to mammals, hence posing a biosafety risk not yet assessed [26][27][28]. Conversely, TarII strains are considered nonpathogenic and have suffered considerable modifications in their kinetoplast DNA (kDNA) due to the constant culture passages through the many years since the first isolation, losing proteins not essential for their life cycle in culture medium [24]. However, the whole genome sequence is available only for TarII strain [29,30]. Therefore, new information on the epidemiology and biology of L. tarentolae, as well as the isolation of new strains are needed to better understand the possible infection in mammals. Overall, this study aimed to evaluate the circulation of L. tarentolae and L. infantum in reptiles, sand flies, and dogs in an endemic area of canine leishmaniasis (CanL) and to obtain new strains of both Leishmania species in axenic cultures.

Ethics statement
Protocols for the collection of reptiles were approved by the ethical committee of the Department of Veterinary Medicine of the University of Bari, Italy (Prot. Uniba 12/20), and authorized by the Ministry for Environment, Land and Sea Protection of Italy (Approval Number 0073267/2019), the Societas Herpetologica Italica and the Istituto Superiore per la Protezione e la Ricerca Ambientale (Approval Number 71216).
Methods and samples are summarized in a flowchart (Fig 1).

Study area
Geckoes, lizards, and sand flies were collected from May to November 2021 in two locations endemic for CanL, near Valenzano municipality (with 4.2 km of distance between each other), Apulia region, Italy [9]. These locations were Campus of Veterinary Medicine, University of Bari "Aldo Moro" (site 1; 41˚1'31.584"N, 16˚54'3.6288"E) and a private owned dog shelter (site 2; 41˚03'04.3"N, 16˚53'39.7"E), where dog blood samples were also collected and screened ( Fig  2). Site 1 had a Mediterranean environment characterized by olive trees, the presence of typical "muretti a secco" (stone walls) where reptiles and sand flies thrive (Fig 3A), while site 2 was a high-walled shelter, with few olive trees where dogs and pigeons were kept ( Fig 3B).

Sample collection
Reptile capturing and processing. Adult reptiles (except for gravid females) were captured by lassoing or by hand, or in the case of snakes using herpetological hooks and sampled before release in the original home range. A small amount of blood (~200μl) obtained via cardiocentesis (in snakes from the ventral coccygeal vein) was used for smears (stained with Diff-Quik), cultivation (~50μl) in a modified Tobie-Evans (TE) medium [31], and the rest was PLOS NEGLECTED TROPICAL DISEASES stored at −20˚C until molecularly processed. Geckoes, positive for L. infantum in blood, were further assessed by bone-marrow biopsy of the femur, according to protocols described elsewhere [32,33]. Briefly, a regular hypodermic needle of adequate size and length (30G X 5/16") was used to collect the sample. The area was prepared aseptically, and the needle was inserted into the bone with continuous and steady pressure and a slight rotational movement. The bone marrow was collected by aspirating the syringe with a slight but steady negative pressure. The obtained material was used for smears in films, cultivation, and DNA extraction.
Entomological survey. During the study period, sand flies were collected twice a week using a sticky trap area of 1 m 2 (32 papers; 21.0 × 29.7 cm) and 4 CDC light traps set from 5:00 p.m. to 8:00 a.m. Collections were carried out during the sand fly activity season [13] until the total absence of sand flies (i.e., three consecutive negative captures). Specimens were stored in 70% ethanol and morphologically identified using taxonomic keys [34]. Alive females collected by CDC traps were dissected with a drop of saline solution and the gut was observed under a microscope to determine the presence of flagellates [20]. The gut content of positive females was cultivated in a modified Tobie-Evans (TE) medium [31].
Dog sampling. Nineteen dogs housed in site 2 were sampled in May and November 2021. A complete physical examination was performed by a veterinarian to assess the health status of the animals. From each dog, whole blood was collected in vacuum containers with K3 EDTA (2.5 ml) and serum collection tubes with clot activator (5 ml).

Serological testing
Dog serum samples were tested by IFAT for the detection of IgG anti-L. infantum as described previously [35]. To evaluate exposure to L. tarentolae, IFAT was performed using promastigotes of L. tarentolae (strain RTAR/IT/81/ISS21-G.6c/LEM124) following the same procedure as for L. infantum IFAT. For both IFAT, serum samples of a dog positive for L. infantum by cytological and molecular analyses, and a healthy dog negative for L. infantum, were used as positive and negative controls, respectively. The samples were scored as positive when they produced clear cytoplasmic and membrane fluorescence of promastigotes from a cut-off dilution of 1:80 [36]. Positive sera were titrated by serial dilutions until negative results were obtained.

Molecular biology
Genomic DNA (gDNA) was extracted from reptile and dog blood samples and cultures, using the GenUPBlood DNA commercial kit (Biotechrabbit GmbH, Hennigsdorf, Germany), according to the manufacturer's instructions. DNA was extracted from bone marrow films using the QIAamp DNA Micro Kit (QIAGEN, Hilden, Germany). gDNA was extracted from the thorax and abdomen (heads and last abdominal segments were removed for morphological identification) of each sand fly female (n = 294) using an in-house method as previously described [37]. All samples (i.e., blood, bone marrow, and sand flies) were tested by duplex real-time PCR (dqPCR) for the detection of L. infantum and L. tarentolae and were considered positive with Ct values up to 38.0 and 38.6, respectively, as described previously [38]. Blood samples were also tested for L. infantum kDNA minicircle (120 bp) by real-time PCR (qPCR), using the protocol described elsewhere [39]. gDNA from L. infantum promastigotes cultured in Tobie-Evans medium from an infected dog living in Italy (zymodeme MON-1) and from L. tarentolae (strain RTAR/IT/81/ISS21-G.6c/LEM124) were used as controls. gDNA extracted from a blood sample from a lizard and a dog tested negative for Leishmania spp. was used as a negative control.
For sequence analyses, culture isolates were amplified by conventional PCR (cPCR) using primers L5.8S/LITSR targeting a partial region of the internal transcribed spacer 1 (ITS1, 300bp) and ran the PCR protocol as described elsewhere [40]. A fragment of the Heat-shock protein 70 gene (hsp70, 1,245 pb) was also amplified using specific primers and the PCR protocol was run as described elsewhere [41].
Engorged sand flies (n = 10) were tested for blood-meal identification by cPCR using primers targeting the vertebrate 16S rRNA gene (600 bp), and the PCR protocol was run as previously described [14]. All PCR reactions consisted of 4 μl of gDNA and 46 μl of PCR mix containing 3 mM MgCl2, 10 mM Tris-HCl (pH 8.3) and 50 mM KCl, 125 μM of each dNTP, 1 pmol/μl of each primer, and 2 U of AmpliTaq Gold (Applied Biosystems, Foster City, CA, USA). The amplified products were examined on 2% agarose gels stained with GelRed (VWR International PBI, Milan, Italy) and visualized on a Gel Logic 100 gel documentation system (Kodak, NY, USA). The amplicons were purified and sequenced in both directions using the same primers as for PCR, employing Big Dye Terminator v.3.1 chemistry in an automated sequencer (3130 Genetic Analyzer, Applied Biosystems, Foster City, CA, USA). All sequences were aligned using Geneious prime software and compared to those available in GenBank using the BLASTn tool (http://blast.ncbi.nlm.nih.gov/blast.cgi).
The genetic relationship of Leishmania species was evaluated using representative hsp70 sequences obtained from culture isolates, and those of reference laboratory strains of L. tarentolae and L. infantum, along with relevant trypanosomatids available in the GenBank database. The phylogenetic tree was inferred using the maximum likelihood (ML) method based on the Kimura 3-parameter model [42], selected by best-fit model analysis, and based on the lowest score obtained by Bayesian Information Criterion (BCI), using MEGA6 software (Kimura, 1980). A discrete Gamma distribution was used to model evolutionary rate differences between sites (4 categories (+G, parameter = 0.1772)). Evolutionary analyses were conducted with 5000 bootstrap replicates using MEGA6 software [43]. The corresponding hsp70 sequence of Trypanosoma brucei gambiense (Accession number: KP208736.1) was used as an outgroup.

Reptile capturing and processing
During the seven-month study period, 37 reptiles of three species (two Hierophis carbonarius snakes, seven Podarcis siculus lizards, and 28 T. mauritanica geckoes) were captured and sampled. In particular, two H. carbonarius, seven P. siculus, 13 T. mauritanica) were collected at site 1, while 15 T. mauritanica were collected at site 2. Leishmania tarentolae was isolated from a T. mauritanica gecko from site 2 ( Table 1). On cytological blood smear examination, trypomastigotes, most likely of Trypanosoma cf. platydactyli, (Fig 4A) were observed in three T. mauritanica geckoes from site 2, whereas amastigote forms of Leishmania spp. were not found in blood. Of the 37 reptile blood samples examined by dqPCR and qPCR, four (10.81%) T. mauritanica geckoes were positive at site 2. Specifically, two geckoes were positive for L. tarentolae, one being the source of the L. tarentolae strain RTAR/IT/21/Ct-25.09, while two other geckoes were positive for L. infantum (one scored positive by dqPCR and one by qPCR; Table 1). Bone marrow films from the two L. infantum PCR positive geckoes revealed amastigote forms within monocytes and macrophages ( Fig 4B; Table 1). Furthermore, DNA extracted from bone marrow films was positive in one gecko (dqPCR Ct-38.6, qPCR Ct-29.07), confirming L. infantum infection (Table 1).

Dog sampling
Of 19 dogs serologically examined, 12 (63.2%) scored positive against promastigotes of L. infantum and L. tarentolae by IFAT in May and 11 (58.8%) in November 2021 (Table 3). Dog blood samples tested by dqPCR and qPCR only yielded positive results for L. infantum in November 2021. Specifically, three dog samples were positive by dqPCR and six samples by qPCR, being three animals positive for both methods (Table 3).

Discussion
Data demonstrate the sympatric circulation of L. infantum and L. tarentolae in geckoes, sand flies, and dogs in an area endemic for CanL. Synanthropic geckoes were found to be PCR positive for L. infantum and Ph. perniciosus for L. tarentolae. Importantly, the finding of L. infantum amastigotes in the bone marrow of geckoes suggests that these animals are not only exposed, but also infected with this Leishmania species. Both reptile species (i.e., P. siculus, T. mauritanica) sampled in this study, have previously been recorded PCR positive to L. infantum and L. tarentolae in the same geographical area, though the prevalence for both L. tarentolae (12.5%) and L. infantum (4.1%) was higher [13]. In the study presented herein, trypanosomatids were only detected in T. mauritanica geckoes probably because it was the main species of reptiles collected. Importantly, sympatric occurrence of these protists (with no co-infection of both Leishmania spp.) was observed in geckoes collected from site 2 (a dog shelter), with a prevalence of 13.3% for both Leishmania spp. and 26.6% for trypomastigotes. Trypanosoma sp. was observed in this study co-infecting three hosts with L. tarentolae, in blood cytology, with different combinations of diagnostic techniques. Though not identified at species level, Trypanosoma cf. platydactyli was previously https://doi.org/10.1371/journal.pntd.0010650.t003 PLOS NEGLECTED TROPICAL DISEASES described (prevalence of 27.5%) co-infecting with L. tarentolae, in T. mauritanica geckoes captured in the surroundings of the Monopoli municipality [31], a city located in the same region (i.e., Apulia) of the present study sites, merely 40 km away. This trypanosomatid has posed an unexpected hindrance for the morphological study of Leishmania spp. in Mediterranean reptiles, being once synonymized with L. tarentolae as their developing forms in culture can be confused (i.e., trypomastigotes may develop into promastigotes after some passages; [22]). Although L. infantum has already been molecularly detected in reptiles [13], this is the first time that amastigote-like forms of this mammalian-associated Leishmania species have been observed in bone marrow aspirate by cytology and further confirmed molecularly. Therefore, these data suggest that reptiles may be infected with L. infantum, further supporting previous studies in which Leishmania spp. pathogenic to mammals were molecularly detected in reptiles [44][45][46]. Accordingly, efforts should be made to isolate L. infantum from reptiles to confirm their role as reservoirs of this medically and veterinary important protist. The detected species composition and abundance of sand flies are in agreement with previous studies in endemic CanL areas [13,14,16,18], where S. minuta was the most abundant species, followed by Ph. perniciosus, the natural vector of L. infantum. The isolation of Leishmania parasites from sand flies confirmed the vector competence of S. minuta for L. tarentolae and represents a new wild type strain for this species [11,31]. Importantly, molecular positivity of S. minuta for L. infantum [13,18,19] and positivity for dog DNA in blood meal [16] may imply a putative role as a vector for this species. However, experimental studies are needed to verify the vector capacity of S. minuta for L. infantum.
The serological results in the studied canine population were consistent with the expected epidemiological scenario, also determined by the molecular detection of only L. infantum in dog blood. Moreover, L. infantum positive blood samples corresponded to animals that had antibody titers higher than 1:640, with lower Ct values in qPCR, which agrees with the higher sensitivity of minicircle qPCR [39]. Indeed, the usefulness of blood qPCR to detect Leishmania is correlated with antibody titration [47], and as high positive predictive value based on clinical evaluation [48]. Nonetheless, given the limited group of dogs selected, discarding a co-infection or a previous exposure to L. tarentolae is difficult. However, this study warrants the high possibility of cross-reactivity seen in most cases of sympatric occurrence of trypanosomatids using IFAT methods [49,50]. Thus, to distinguish the infecting species of Leishmania, specific confirmatory serological tests should be developed.
This study also represents the most recent isolation of L. tarentolae strains from both reptiles and sand flies, thus not suffering from genetic drift due to the long-term cultivation. Although the ITS1 and hsp70 sequences were highly similar to those of the reference strains, such new strains may represent transiently infectious parasites that should be further molecularly characterized and subjected to phagocytosis and in vitro infectivity analyses [27,29]. Indeed, further attempts should be made to isolate L. tarentolae from other hosts (i.e., dogs and humans) to fully unravel the infection in mammalian hosts.
The epidemiological scenarios of canine leishmaniasis in the Mediterranean basin can change depending on multiple factors, such as geographic area, sand fly species, and/or canine populations. The scenario observed in the periurban areas of southern Italy adds to this factor the presence of often ignored sand fly, reptile, and Leishmania species, namely S. minuta, T. mauritanica, and L. tarentolae. Synanthropic omnipresent geckoes are exposed not only to their specific Leishmania species, but also to L. infantum, which can develop into amastigotes in the bone marrow. The studied sand fly species display different levels of anthropophilic feeding behavior. The isolation of new L. tarentolae strains may provide new information on transient infection, similar to the LEM125 strains. Confirming the isolation of L. infantum from reptiles and L. tarentolae from mammals is needed to unravel the epidemiological context given by the sympatric occurrence of both Leishmania species.